Molecular dissection of the genes involved in biosynthesis of polyketide and secondary metabolites of Aspergillus fumigatus and their significance in virulence. Aspergillus is one of the most common fungal pathogens affecting neutropenic patients and other types of immunocompromised individuals such as those with Chronic Granulomatous Disease of Childhood. Among a dozen species of Aspergillus reported to cause infection in humans, A. fumigatus is the most common species reported to cause invasive aspergillosis. All Aspergillus species propagate by conidia (spores), which humans encounter daily through inhalation. During the previous years, we have focused our attention on the molecular genetic aspects of conidial pigment biosynthesis since pigment is one of the visible components of the wall that protect conidia. We have characterized six developmentally associated genes involved in pentaketide melanin synthesis which are clustered within a 19kb fragment of A. fumigatus genomic DNA. Furthermore, we have shown that the conidial pigment synthetic pathway plays an important role in pathogenesis. During 2004-2005, we optimized the Agrobacterium tumefaciens mediated transformation (ATMT) system for A. fumigatus. ATMT has proven to be an efficient molecular tool for insertional mutagenesis as well as gene disruption by homologous recombination.[unreadable] Using the ATMT system, we disrupted the LaeA gene known to be a global regulator for the expression of several important toxins among which gliotoxin is considered to be most important for virulence of A. fumigatus. Unlike the previous report on LaeA disruptant by the spheroplast method, the LaeA gene disrupted via ATMT was indistinguishable from the wild type in its mycelial pigment formation. During 2005-2006, we have studied the effect of laeA deletion in the ability of Aspergillus culture supernatant to cause cell detachment and death of EL4 thymoma and MEF cell lines, inhibition of oxidative burst in human PMN, morphogenesis of conidia and virulence in mouse strain 129S. We also deleted the glip gene which encodes the non-ribosomal peptide synthase catalyzing the first step of gliotoxin production. The laeA ko strain, unlike previous report, produced conidia morphologically indistinguishable from those produced by the wild type strain. Culture filtrates of laeA ko strain contained 85% less gliotoxin than that of wild type according to HPLC analysis and the level of gliotoxin in the culture supernatant of laeA reconstituted strain was similar to that of wild type. The laeA strain was significantly less virulent than the wild type or the laeA reconstituted strain for 129S mice and the progression of pneumonia was considerably slower in mice infected with laeA ko strain compared to the wild type. The culture supernatant of the laeA ko strain failed to block oxidative burst of human nutrophils (PMN) while the supernatant from the wild type and complemented strains in the same concentration inhibited the oxidative burst. Furthermore, culture filtrates of wild type and laeA complemented strains were several fold more potent in causing cell detachment and apoptosis in both cell lines of EL4 thymoma and murine embryonic fibroblasts (MEF). These findings indicated that the laeA gene regulates secondary metabolites such as gliotoxin but not morphogenesis in A. fumigatus.